Oligonucleotide-based inhibitors comprising locked nucleic acid motif

ABSTRACT

The present invention relates to chemical modification motifs for oligonucleotides. The oligonucleotides of the present invention, such as chemically modified antisense oligonucleotides, can have increased in vivo efficacy. The chemically modified oligonucleotides provide advantages in one or more of potency, efficiency of delivery, target specificity, toxicity, and/or stability. The chemically modified oligonucleotides have a specific chemical modification motif or pattern of locked nucleic acids (LNAs). The oligonucleotide (e.g. antisense oligonucleotide) can target RNA, such as miRNA or niflNA. Also provided herein are compositions comprising the chemically modified oligonucleotides and methods of using the chemically modified oligonucleotides as therapeutics for various disorders, including cardiovascular disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/662,746, filed Jun. 21, 2012, and U.S. Provisional Application No.61/801,533, filed Mar. 15, 2013, both of which are hereby incorporatedby reference in their entireties.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:MIRG_(—)036_(—)02US_SeqList_ST25.txt, date recorded Jun. 21, 2013, filesize 85 kilobytes).

FIELD OF THE INVENTION

The present invention relates to chemical modification motifs foroligonucleotides, such as antisense oligonucleotides, including mRNA andmicroRNA (miRNA or miR) inhibitors. The oligonucleotides of the presentinvention, such as chemically modified antisense oligonucleotides, forexample, miRNA antisense oligonucleotides, can have advantages inpotency, efficiency of delivery, target specificity, stability, and/ortoxicity when administered to a subject.

BACKGROUND OF THE INVENTION

Delivery of oligonucleotides to the body, such as an antisense-basedtherapeutics, poses several challenges. The binding affinity andspecificity to a target, efficiency of cellular uptake, and nucleaseresistance are all factors in the delivery and activity of anoligonucleotide-based therapeutic. For example, when oligonucleotidesare introduced into intact cells they are attacked and degraded bynucleases leading to a loss of activity. Thus, a useful oligonucleotideshould have good resistance to extra- and intracellular nucleases, aswell as be able to penetrate the cell membrane.

Polynucleotide analogues have been prepared in an attempt to avoid theirdegradation, e.g. by means of 2′ substitutions (Sproat et al, Nucleic.Acids Research 17 (1989), 3373-3386). However, such modifications oftenaffect the polynucleotide's potency for its intended biological action.Such reduced potency may be due to an inability of the modifiedpolynucleotide to form a stable duplex with the target RNA and/or a lossof interaction with the cellular machinery. Other modifications includethe use of locked nucleic acids, which has the potential to improveRNA-binding affinity (Veedu and Wengel, RNA Biology 6: 3, 321-323(2009)), however, in vivo efficacy can be low. An oligonucleotide usedas an antisense therapeutic should have high affinity for its target toefficiently impair the function of its target (such as inhibitingtranslation of a mRNA target, or inhibiting the activity of a miRNAtarget). However, modification of oligonucleotides can decrease itsaffinity and binding specificity, as well as its ability to impair thefunction of its target.

Thus, despite the variety of methods described for the delivery ofoligonucleotides as a therapeutic, there is a need for improved chemicalmodifications for stable and efficacious oligonucleotide-basedinhibitors.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that aspecific chemical modification pattern or motif of an oligonucleotidecan increase the potency, efficiency of delivery, target specificity,stability, and/or improve the toxicity profile when administered to asubject. The present inventors have discovered specific oligonucleotidechemistry modification patterns or motifs with the potential to improvethe delivery, stability, potency, specificity, and/or toxicity profileof the oligonucleotide. For example, oligonucleotide chemistrymodification patterns or motifs for miRNA inhibitors can improve thedelivery, stability, potency, specificity, and/or toxicity profile ofthe miRNA inhibitor, thus, effectively targeting miRNA function in atherapeutic context.

The present invention provides oligonucleotides with a chemistrymodification pattern or motif capable of inhibiting the expression(e.g., abundance) of miRNA with improved properties, such as increasedin vivo efficacy. This chemistry modification pattern or motif can beapplied to other oligonucleotides for targeting other therapeutictargets, such as mRNA. Thus, the present invention provides a noveltherapeutic for the treatment of a variety of diseases, includingcardiovascular diseases, obesity, diabetes, and other metabolicdisorders.

The oligonucleotide with the specific chemical modification pattern ormotif can have an increased in vivo efficacy as compared to anoligonucleotide with the same nucleotide sequence but different chemicalmodification pattern or motif. For example, an oligonucleotide with aspecific locked nucleic acid (LNA) pattern can have an increased in vivoefficacy as compared to an oligonucleotide with the same nucleotidesequence but different LNA pattern.

In one embodiment, the oligonucleotide of the present inventioncomprises a sequence complementary to the seed region of a miRNA,wherein the sequence comprises at least five LNAs. The oligonucleotidecan comprise at least five LNAs complementary to the seed region of amiRNA and at least one non-locked nucleotide. In some embodiments, thenon-locked nucleotide is in a region that is complementary to the seedregion. The oligonucleotide can have increased in vivo efficacy ascompared to a second oligonucleotide comprising the same sequence andLNA composition and different LNA motif. The oligonucleotide cancomprise a LNA at the 5′ end, 3′ end, or both 5′ and 3′ ends. In someembodiments, the oligonucleotide comprises three or fewer contiguousLNAs. For example, the oligonucleotide comprises no more than threecontiguous LNAs. The oligonucleotide can be at least 16 nucleotides inlength. In some embodiments, the oligonucleotide can be from 8 to 20nucleotides in length, from 18 to 50 nucleotides in length, from 10 to18 nucleotides in length, or from 11 to 16 nucleotides in length. Theoligonucleotide in some embodiments is about 8, about 9, about 10, about11, about 12, about 13, about 14, about 15, about 16, about 17, or about18 nucleotides in length.

In another embodiment, the oligonucleotide of the present inventioncomprises a sequence of 16 nucleotides, wherein the sequence comprisesat least five LNAs, a LNA at the 5′ end, a LNA at the 3′ end, and nomore than three contiguous LNAs. The oligonucleotide, from the 5′ end tothe 3′ end, can comprise LNAs at positions 1, 5, 6, 8, 10, 11, 13, 15,and 16 of the sequence.

The oligonucleotide described herein can comprise one or more non-lockednucleotides. In some embodiments, at least one of the non-lockednucleotides is 2′ deoxy, 2′ O-alkyl or 2′ halo. In another embodiment,all of the non-locked nucleotides are 2′ deoxy, 2′ O-alkyl, 2′ halo, orany combination thereof.

In some embodiments, the oligonucleotide described herein comprises atleast one LNA with a 2′ to 4′ methylene bridge. The oligonucleotide canhave a 5′ cap structure, 3′ cap structure, or 5′ and 3′ cap structure.In some embodiments, the oligonucleotide comprises one or morephosphorothioate linkages or is fully phosphorothioate-linked. Theoligonucleotide can have one to three phosphate linkages. Theoligonucleotide can further comprise a pendent lipophilic or hydrophilicgroup.

In one embodiment, the oligonucleotide is an inhibitor of a RNA, such asan inhibitor of its expression or activity. In one embodiment, theoligonucleotide is a miRNA inhibitor. For example, the oligonucleotidecan comprise a sequence that is substantially or completelycomplementary to a nucleotide sequence of a miRNA or fragment thereof.The miRNA can be expressed in any tissue, or selectively expressed in atissue. In one embodiment, the tissue is cardiac tissue. For example,the miRNA is selectively expressed in cardiac tissue.

The oligonucleotide can be an inhibitor of any miRNA. In someembodiments, the oligonucleotide can be an inhibitor of any miRNA, butnot miR-208a, miR-208b, or miR-499. Such inhibitors are described in,for example. International Publication No. WO 2012/083005, which ishereby incorporated by reference in its entirety. In one embodiment, theoligonucleotide is an inhibitor of a miR selected from Table 1 or Table2. In yet another embodiment, the oligonucleotide is an inhibitor ofmiR-15 a, miR-15b, miR-16-1, miR-16-2, miR-24, miR-25, miR-26a, miR-497,miR-195, miR-424, a let 7 family member, miR-21, miR-199a-b, miR-214,miR-10a-b, miR-16, miR-125b, miR-146a-b, miR-221, miR-222, a miR-30family member, miR-126, miR-133, miR-1, miR-143, miR-145, miR-486,miR-92a, miR-320, miR-1-1, miR-1-2, miR-451, miR-378, miR-378*, miR-92,miR-34a, miR-34b, miR-34c, miR-29, ormiR-33.

In yet another embodiment, the oligonucleotide can be an inhibitor ofmRNA. For example, the sequence can be substantially or completelycomplementary to a nucleotide sequence of an mRNA or fragment thereof.

Also provided herein is a pharmaceutical composition comprising aneffective amount of the oligonucleotide described herein, or apharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable carrier or diluent. In some embodiments, thepharmaceutically-acceptable carrier can comprise a colloidal dispersionsystem, macromolecular complex, nanocapsule, nanoparticle, microsphere,bead, oil-in-water emulsion, micelle, mixed micelle, or liposome. Inanother embodiment, the pharmaceutically-acceptable carrier or diluentconsists essentially of saline.

The present invention also provides methods of producing and using anoligonucleotide described herein. A method of reducing or inhibitingactivity of a miRNA in a cell comprising contacting the cell with anoligonucleotide described herein is also provided. Also disclosed hereinis a method of reducing expression of an mRNA in a cell comprisingcontacting the cell with an oligonucleotide disclosed herein. The cellcan be any cell type, such as a heart cell. The cell can be in vivo orex vivo. In one embodiment, the cell is a mammalian cell.

A method of preventing or treating a condition in a subject associatedwith or mediated by expression of an RNA is also provided. The methodcan comprise administering to the subject a pharmaceutical compositioncomprising an oligonucleotide disclosed herein. In one embodiment, amethod of preventing or treating a condition in a subject associatedwith or mediated by the activity of a miRNA comprises administering tothe subject a pharmaceutical composition comprising the oligonucleotidedisclosed herein. In another embodiment, a method of preventing ortreating a condition in a subject associated with or mediated by theactivity of a mRNA comprises administering to the subject apharmaceutical composition comprising the oligonucleotide disclosedherein. The condition can be a heart condition, such as pathologiccardiac hypertrophy, myocardial infarction, myocardial ischemia,ischemia-reperfusion injury, cardiomyopathy, or heart failure. Thepharmaceutical composition can be administered by parenteraladministration, such as by intravenous, subcutaneous, intraperitoneal,or intramuscular administration. In some embodiments, administration isby direct injection into cardiac tissue. In yet in some embodiments, thecomposition is administered by oral, transdermal, sustained release,controlled release, delayed release, suppository, catheter, orsublingual administration. Furthermore, the subject can be a human. Insome embodiments, an oligonucleotide disclosed herein is delivered at adose of between about 10 mg/kg to about 100 mg/kg, between about 10mg/kgto about 50 mg/kg, between about 10 mg/kg to about 25 mg/kg. In someembodiments, an oligonucleotide disclosed herein is delivered at a doseof about 100 mg/kg or less, about 50 mg/kg or less, about 25 mg/kg orless, or about 10 mg/kg or less. In one embodiment, the oligonucleotideis formulated in saline and administered subcutaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Location of LNA and DNA bases for 16 antimiRs designed totarget miR-208a (SEQ ID NOs: 76-91). LNA bases are represented by acapital letter. DNA bases are represented by a lower case letter. (B)MiR-208a inhibition by antimiR-208a compounds. All antimiR compoundsshowed significant inhibition in the left ventricle. #p<0.05 vs Saline.*p<0.05 vs. control oiigo, M-10591. (C) Real-time PCR from cardiactissue of antimiR-208a-treated rats showed differing targetde-repression in vivo using Dynlt1 as a primary readout for efficacy andtarget de-repression. #p<0.05 vs Saline. *p<0.05 vs. control oiigo,M-10591. (D) Serum levels of toxicology parameters. Four days afterinjection, plasma was collected from all groups. No antimiR-208aoligonucleotide or control oligonucleotide showed increased levels ofliver toxicity as assessed by ALT and AST measurements, or kidneytoxicity as assessed by BUN measurements compared to saline controls.(E) Quantification of antimiR from heart, liver, and kidney four daysafter a single 25 mg/kg subcutaneous dose. Distribution to the heart ismuch lower than liver and kidney. Efficacious compounds are not morerobustly distributed to the heart.

FIG. 2. (A) Location of LNA and DNA bases for 9 antimiRs designed totarget miR-208b (SEQ ID NOs: 92-100). LNA bases are represented by acapital letter. DNA bases are represented by a lower case letter. (B)MiR-208b inhibition by antimiR-208b compounds. All antimiR compoundsshowed significant miR-208b inhibition in the left ventricle. (C)Real-time PCR from cardiac tissue of antimiR-208b treated rats showeddiffering target de-repression in vivo using Dynlt1 as a primary readoutfor efficacy and target de-repression. *p<0.05 vs. Saline

FIG. 3. Silencing. (A) Location of LNA and DNA bases for 7 antimiRsdesigned to target miR-378 (SEQ ID NOs: 101-107). LNA bases arerepresented by a capital letter. DNA bases are represented by a lowercase letter. (B) MiR-378 inhibition by antimiR-378 compounds. AllantimiR compounds showed significant miR-378 inhibition in the leftventricle. (C) Real-time PCR from cardiac tissue of antimiR-378 treatedrats showed differing target de-repression in vivo using Gfpt2 as aprimary readout for efficacy and target de-repression. #p<0.05 vs.Saline

FIG. 4, (A) Location of LNA and DNA bases for 7 antimiRs designed totarget miR-29 (SEQ ID NOs: 108-114). LNA bases are represented by acapital letter. DNA bases are represented by a lower case letter. (B)MiR-29 family inhibition by antimiR-29 compounds in heart (top panel),liver (middle panel), and kidney (bottom panel). All antimiR compoundsshowed significant miR-29 family inhibition in heart, liver, and kidney,(C) Real-time PCR from heart (top panel), liver (middle panel), andkidney (bottom panel) of antimiR-29 treated rats showed differing targetde-repression in vivo using Dnmt3b and Mcll as a primary readout forefficacy and target de-repression. *p<0.05 vs. Saline; #p<0.05 vs.Control oligonucleotide M-10591. (D) Quantification of antimiR compoundsfrom heart and liver four days after a single 25 mg/kg subcutaneousdose. Distribution to the heart is much lower than liver. Moreefficacious compounds are not more robustly distributed to the heartcompared to less efficacious compounds.

FIG. 5. (A) Location of LNA and DNA bases for 5 antimiRs designed totarget miR-199a (SEQ ID NOs: 115-119). LNA bases are represented by acapital letter. DNA bases are represented by a lower case letter. (B)MiR-199a inhibition by antimiR-199 compounds in heart, lung, liver (Li),and kidney (K). All antimiR compounds showed significant miR-199ainhibition in heart, lung, liver, and kidney. (C) Real-time PGR fromheart, lung, liver (Li), and kidney (K) of antimiR-199-treated ratsshowed differing target de-repression in vivo using Ddrl as a primaryreadout for efficacy and target de-repression. M-10518 consistentlyappeared to show target de-repression across multiple tissues. *p<0.05vs. Saline.

FIG. 6. Real-time PGR from endothelial cells isolated from heart tissueof antimiR-92a-treated rats showed differing target de-repression invivo using Map2K4 as a primary readout for efficacy and targetde-repression. *p<0.05 vs. Saline.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that aspecific chemical modification pattern or motif of an oligonucleotidecan improve the potency, efficiency of delivery, target specificity,stability, and/or toxicity when administered to a subject. Theoligonucleotide with the specific chemical modification pattern or motifcan have an increased in vivo efficacy as compared to an oligonucleotidewith the same nucleotide sequence but different chemical modificationpattern or motif. For example, an oligonucleotide with a specificLNA/DNA pattern can have an increased in vivo efficacy as compared to anoligonucleotide with the same nucleotide sequence but different LNA/DNApattern.

The invention provides in some embodiments, oligonucleotides capable ofinhibiting, in a specific fashion, the expression or abundance of an RNAspecies, such as a miRNA or mRNA. The invention further providespharmaceutical compositions comprising the oligonucleotides, and methodsof treating patients having conditions or disorders relating to orinvolving the RNA, such as miRNA or mRNA, such as a variouscardiovascular conditions. In various embodiments, the oligonucleotidesprovide advantages in one or more of potency, efficiency of delivery,target specificity, toxicity, and/or stability.

In one aspect, the present invention provides an oligonucleotide capableof reducing the expression or abundance of an RNA, such as a mRNA ormiRNA. The oligonucleotide of the present invention may have increasedin vivo efficacy as compared to another oligonucleotide with the samenucleotide sequence but different chemical modification motif orpattern. For example, a first and a second oligonucleotide each have thesame nucleotide sequence that targets a miRNA. The first oligonucleotidehas a chemical modification motif or pattern that differs from thesecond oligonucleotide. Both the first and second oligonucleotides arecapable of reducing the expression or abundance of a miRNA, However, thefirst oligonucleotide with a first chemical modification motif has ahigher in vivo efficacy as compared to the second oligonucleotide with adifferent chemical modification motif, as measured by the amount ofde-repression of one or more of the miRNA's targets.

The activity of the oligonucleotide in reducing the expression orabundance of an RNA species, such as miRNA., may be determined in vitroand/or in vivo. For example, when inhibition of a miRNA activity isdetermined in vitro, the activity may be determined using a dualluciferase assay, such as that described herein. The oligonucleotidesignificantly inhibits such activity, as determined in the dualluciferase activity, at a concentration of about 50 nM or less, or inother embodiments, 40 nM or less, 20 nM or less, or 10 nM or less. Forexample, the oligonucleotide may have an IC50 for inhibition of a miRNAactivity of about 50 nM or less, about 40 nM or less, about 30 nM orless, or about 20 nM or less, as determined in the dual luciferaseassay. The dual luciferase assay, as exemplified by the commerciallyavailable product PsiCHECK™ (Promega), involves placement of the miRrecognition site in the 3′ UTR of a gene for a detectable protein (e.g.,renilla luciferase). The construct is co-expressed with the targetmiRNA, such that inhibitor activity can be determined by change insignal. A second gene encoding a detectable protein (e.g., fireflyluciferase) can be included on the same plasmid, and the ratio ofsignals determined as an indication of antimiR activity.

Alternatively, or in addition, the activity of the oligonucleotide inreducing the expression or abundance of an RNA species, such as miRNA,may be determined in a suitable mouse or rat model, such as thosedescribed herein, where inhibition (e.g., by at least 50%) of a miRNA isobserved at an oligonucleotide dose, such as a dose of about 50 mg/kg orless, about 25 mg/kg or less, about 10 mg/kg or less or about 5 mg/kg orless. In some embodiments, the activity of the oligonucleotide isdetermined in an animal model, such as described in WO 2008/016924,which descriptions are hereby incorporated by reference. For example,the oligonucleotide may exhibit at least 50% target miRNA inhibition,such as a dose of about 50 mg/kg or less, about 25 mg/kg or less, suchas about 10 mg/kg or less or about 5 mg/kg or less. In such embodiments,the oligonucleotide may be dosed intravenously or subcutaneously tomice, and the oligonucleotide may be formulated in saline.

The in vivo efficacy of the oligonucleotide may be determined byassessing the level or amount of de-repression of one or more of themiRNA's targets in a suitable mouse or rat model, such as thosedescribed herein. The oligonucleotide may exhibit at least 50% targetde-repression at a dose of about 50 mg/kg or less, about 25 mg/kg orless, about 10 mg/kg or less or about 5 mg/kg or less. In suchembodiments, the oligonucleotide may be dosed intravenously orsubcutaneously to mice, and the oligonucleotide may be formulated insaline.

In these or other embodiments, the oligonucleotides of the presentinvention can be stable after administration, being detectable in thecirculation and/or target organ for at least three weeks, at least fourweeks, at least five weeks, or at least six weeks, or more, followingadministration. Thus, the oligonucleotides of the present invention mayprovide for less frequent administration, lower doses, and/or longerduration of therapeutic effect.

The nucleotide sequence of the oligonucleotide can be substantiallycomplementary to a nucleotide sequence of a RNA, such as a mRNA ormiRNA. In some embodiments, the miRNA is not miR-208a, miR-208b, ormiR-499. The oligonucleotide comprises at least one LNA, such as atleast five, at least seven or at least nine LNAs. In some embodiments,the oligonucleotide comprises a mix of LNA and non-locked nucleotides.For example, the oligonucleotide may contain at least five or at leastseven or at least nine locked nucleotides, and at least one non-lockednucleotide.

Generally, the length of the oligonucleotide and number and position oflocked nucleotides is such that the oligonucleotide reduces RNAexpression or abundance, such as mRNA expression or miRNA expression, atan oligonucleotide concentration of about 50 nM or less in the in vitroluciferase assay, or at a dose of about 50 mg/kg or less, or about 25mg/kg or less in a suitable mouse or rat model, each as describedherein. In some embodiments, the oligonucleotide is a miRNA inhibitor,such that the length of the oligonucleotide and number and position oflocked nucleotides is such that the oligonucleotide reduces miRNAactivity as determined by target de-repression, at a dose of about 50mg/kg or less, or about 25 mg/kg or less in a suitable mouse or ratmodel, such as those described herein.

The oligonucleotide of the present invention can comprise a sequence ofnucleotides in which the sequence comprises at least five LNAs, a LNA atthe 5′ end of the sequence, a LNA at the 3′ end of the sequence, or anycombination thereof. In one embodiment, the oligonucleotide comprises asequence of nucleotides in which the sequence comprises at least fiveLNAs, a LNA at the 5′ end of the sequence, a LNA at the 3′ end of thesequence, or any combination thereof, wherein three or fewer of thenucleotides are contiguous LNAs. For example, the oligonucleotidecomprises no more than three contiguous LNAs. For example, theoligonucleotide may comprise a sequence with at least five LNAs, a LNAat the 5′ end, a LNA at the 3′ end, and no more than three contiguousLNAs. The oligonucleotide may comprise a sequence with at least fiveLNAs, a LNA at the 5′ end, a LNA at the 3′ end, and no more than threecontiguous LNAs, wherein the sequence is at least 16 nucleotides inlength. The sequence can be substantially or completely complementary toa RNA, such as mRNA, or miRNA., wherein a substantially complementarysequence may have from 1 to 4 mismatches (e.g., 1 or 2mismatches) withrespect to its target sequence. In one embodiment, the target sequenceis a miRNA, such that the oligonucleotide is a miRNA inhibitor, orantimiR. The miRNA can be any miRNA, such as, but not limited to, thoselisted in Table 1 or Table 2. Exemplary miRNA therapeutic utilities aredisclosed in the US and PCT patent references listed in Table 2 below,each of which is hereby incorporated by reference in its entirety. Themature and pre-processed forms of miRNAs are disclosed in the patentreferences listed in Table 2, and such descriptions are also herebyincorporated by reference.

TABLE 1  miRNA miRNA Sequence SEQ ID NO:  1 UGGAAUGUAAAGAAGUAUGUAU 1100  AACCCGUAGAUCCGAACUUGUG 2  10a UACCCUGUAGAUCCGAAUUUGUG 3  10bUACCCUGUAGAACCGAAUUUGUG 4 125b UCCCUGAGACCCUAACUUGUGA 5 126 UCGUACCGUGAGUAAUAAUGCG 6 128  UCACAGUGAACCGGUCUCUUU 7 133aUUUGGUCCCCUUCAACCAGCUG 8 133b UUUGGUCCCCUUCAACCAGCUA 9 139 UCUACAGUGCACGUGUCUCCAG 10 143  UGAGAUGAAGCACUGUAGCUC 11 145 GUCCAGUUUUCCCAGGAAUCCCU 12 146a UGAGAACUGAAUUCCAUGGGUU 13 146bUGAGAACUGAAUUCCAUAGGCU 14 150  UCUCCCAACCCUUGUACCAGUG 15  15aUAGCAGCACAUAAUGGUUUGUG 16  15b UAGCAGCACAUCAUGGUUUACA 17 16UAGCAGCACGUAAAUAUUGGCG 18 181b AACAUUCAUUGCUGUCGGUGGGU 19 195 UAGCAGCACAGAAAUAUUGGC 20 197  UUCACCACCUUCUCCACCCAGC 21 199aCCCAGUGUUCAGACUACCUGUUC 22 199b-5p CCCAGUGUUUAGACUAUCUGUUC 23 199b-3pACAGUAGUCUGCACAUUGGUUA 24 208a AUAAGACGAGCAAAAAGCUUGU 25 208bAUAAGACGAACAAAAGGUUUGU 26  20a UAAAGUGCUUAUAGUGCAGGUAG 27 21UAGCUUAUCAGACUGAUGUUGA 28 214  ACAGCAGGCACAGACAGGCAGU 29 22AAGCUGCCAGUUGAAGAACUGU 30 221  AGCUACAUUGUCUGCUGGGUUUC 31 222 AGCUACAUCUGGCUACUGGGU 32 224  CAAGUCACUAGUGGUUCCGUU 33  23aAUCACAUUGCCAGGGAUUUCC 34 24 UGGCUCAGUUCAGCAGGAACAG 35 25CAUUGCACUUGUCUCGGUCUGA 36  26a UUCAAGUAAUCCAGGAUAGGCU 37  26bUUCAAGUAAUUCAGGAUAGGU 38 28 AAGGAGCUCACAGUCUAUUGAG 39  29aUAGCACCAUCUGAAAUCGGUUA 40  29b UAGCACCAUUUGAAAUCAGUGUU 41  29cUAGCACCAUUUGAAAUCGGUUA 42  30a UGUAAACAUCCUCGACUGGAAG 43  30bUGUAAACAUCCUACACUCAGCU 44  30c UGUAAACAUCCUACACUCUCAGC 45  30dUGUAAACAUCCCCGACUGGAAG 16  30e UGUAAACAUCCUUGACUGGAAG 47  33aGUGCAUUGUAGUUGCAUUGCA 48  33b GUGCAUUGCUGUUGCAUUGC 49  34aUGGCAGUGUCUUAGCUGGUUGU 50  34b CAAUCACUAACUCCACUGCCAU 51  34cAGGCAGUGUAGUUAGCUGAUUGC 52 320  AAAAGCUGGGUUGAGAGGGCGA 53 342-3pUCUCACACAGAAAUCGCACCCGU 54 382  GAAGUUGUUCGUGGUGGAUUCG 55 422aACUGGACUUAGGGUCAGAAGGC 56 378  ACUGGACUUGGAGUCAGAAGG 57 378*CUCCUGACUCCAGGUCCUGUGU 58 424  CAGCAGCAAUUCAUGUUUUGAA 59 451 AAACCGUUACCAUUACUGAGUU 60 483-3p UCACUCCUCUCCUCCCGUCUU 61 484 UCAGGCUCAGUCCCCUCCCGAU 62 486-5p UCCUGUACUGAGCUGCCCCGAG 63 497 CAGCAGCACACUGUGGUUUGU 64 499  UUAAGACUUGCAGUGAUGUUU 65 542-5pUCGGGGAUCAUCAUGUCACGAGA 66  92a UAUUGCACUUGUCCCGGCCUGU 67  92bUAUUGCACUCGUCCCGGCCUCC 68 let-7a UGAGGUAGUAGGUUGUAUAGUU 69 let-7bUGAGGUAGUAGGUUGUGUGGUU 70 tet-7c UGAGGUAGUAGGUUGUAUGGUU 71 let-7dAGAGGUAGUAGGUUGCAUAGUU 72 let-7e UGAGGUAGGAGGUUGUAUAGUU 73 let-7fUGAGGUAGUAGAUUGUAUAGUU 74 let-7g UGAGGUAGUAGUUUGUACAGUU 75

TABLE 2 miRNA Indications Reference miR-208a/miR-208b/miR- Pathologiccardiac hypertrophy, WO 2008/016924 (208a) 499 myocardial infarction,heart failure WO 2009/018492 (208b/499) miR-208a/miR-208b MetabolicDisorders (obesity, PCT/US2012/059349, filed Oct. 9, hyperlipidemia,diabetes, metabolic 2012 syndrome, hypercholesterolemia; hepaticsteatosis) miR-15/miR-16/miR-195 Pathologic cardiac hypertrophy, WO2009/062169 myocardial infarction, heart failure miR-29 Profibroticagents to convert soft plaques WO 2009/018493 (vulnerable plaques) tofibrotic tissue; induce collagen deposition miR-126 Pathologicvascularization WO 2010/019574 miR-145 Muscle injury WO 2007/070483miR-1/miR-133 Muscle injury (antagonist/agonist of each WO 2007/070483miRNA applied in combination at different times) miR-451 Polycythemia WO2012/148373 miR-378/miR-378* Metabolic disorders (obesity, WO2011/153542 hyperlipidemia, diabetes, metabolic syndrome,hypercholesterolemia; hepatic steatosis); Pathologic cardiachypertrophy, myocardial infarction, heart failure miR-92 Promotesangiogenesis and vessel repair US 2010/0324118 A1 miR-34a Myocardialinfarction US 2012/0238619 A1 miR-145 Pulmonary arterial hypertension WO2012/153135 miR-33 Statin-induced hepatotoxicity, US 20110281933 A1cholestasis, increasing HDL cholesterol

In some embodiments, the oligonucleotide comprises a sequence that issubstantially or completely complementary to a miRNA that is selectedfrom the group consisting of, but not limited to: miR-15a, miR-15b.miR-16-1, miR-16-2, miR-24, miR-25, miR-26a, miR-497, miR-195, miR-424,a let 7 family member, miR-21, miR-199a-b, miR-214, miR-10a-b, miR-16,miR-125b, miR-146a-b, miR-221, miR-222, a miR-30 family member, miR-126,miR-133, miR-1, miR-143, miR-145, miR-486, miR-92a, miR-320, miR-1-1,miR-1-2, miR-451, miR-378, miR-378*. miR-92, miR-34a, miR-34b, miR-34c,miR-29, or miR-33. In some embodiments, the miRNA is not miR208a,miR208b, or miR-499, such as described in International Publication No.WO 2012/083005, which is hereby incorporated by reference in itsentirety. In some embodiments, the miRNA is expressed in a specifictissue, such as kidney, liver, or cardiac tissue. In yet anotherembodiment, the miRNA is selectively expressed in a tissue, such askidney, liver, or cardiac tissue.

In yet another embodiment, the oligonucleotide of the present inventioncan comprise a sequence complementary to the seed region of a miRNA,wherein the sequence comprises at least five LNAs. The “seed region of amiRNA” is the portion spanning bases 2 to 9 at the 5′ end of the miRNA.The miRNA can be any miRNA, such as, but not limited to those listed inTable 1 or Table 2. The miRNA can be, but is not limited to: miR-1.5a,miR-15b, miR-16-1, miR-16-2, miR-24, miR-25, miR-26a, miR-497, miR-195,miR-424, a let 7 family member, miR-21, miR-199a-b, miR-214, miR-10a-b,miR-16, miR-125b, miR-146a-b, miR-221, miR-222, a miR-30 family member,miR-126, miR-.133, miR-1, miR-143, miR-145, miR-486, miR-92a, miR-320,miR-1-1, miR-1-2, miR-451, miR-378, miR-378*, miR-92, miR-34a, miR-34b,miR-34c, miR-29, or miR-33. In some embodiments, the miRNA is notmiR208a, miR208b, or miR-499. The sequence can be substantially orcompletely complementary to the miRNA. In some embodiments, the miRNA isexpressed in a specific tissue, such as kidney, liver, or cardiactissue. In yet another embodiment, the miRNA is selectively expressed ina tissue, such as kidney, liver, or cardiac tissue. In some embodiments,the miRNA is selectively expressed in a particular cell type, including,but not limited to, cardiomyocytes, myocytes, fibroblasts, smooth musclecells, endothelial cells, and monocytes.

The oligonucleotide comprising a sequence complementary to the seedregion of a miRNA, wherein the sequence comprises at least five LNAs,may comprise a LNA at the 5′ end or a LNA at the 3′ end, or both a LNAat the 5′ end and 3′ end. In one embodiment, the oligonucleotidecomprising at least 5 LNAs, a LNA at the 5′ end and/or a LNA at the 3′end, also has three or fewer consecutive LNAs. In some embodiments, thesequence is at least 16 nucleotides in length. The sequencecomplementary to the seed region of a miRNA can be substantiallycomplementary or completely complementary.

The oligonucleotide of the present invention contains one or more lockednucleic acid (LNAs) residues, or “locked nucleotides.” LNAs aredescribed, for example, in U.S. Pat. Nos. 6,268,490; 6,316,198;6,403,566; 6,770,748; 6,998,484; 6,670,461; and 7,034,133, all of whichare hereby incorporated by reference in their entireties. LNAs aremodified nucleotides or ribonucleotides that contain an extra bridgebetween the 2′ and 4′ carbons of the ribose sugar moiety resulting in a“locked” conformation, and/or bicyclic structure. In one embodiment, theoligonucleotide contains one or more LNAs having the structure shown bystructure A below. Alternatively or in addition, the oligonucleotide maycontain one or more LNAs having the structure shown by structure Bbelow. Alternatively or in addition, the oligonucleotide contains one ormore LNAs having the structure shown by structure C below.

Other suitable locked nucleotides that can be incorporated in theoligonucleotides of the present invention include those described inU.S. Pat. Nos. 6,403,566 and 6,833,361, both of which are herebyincorporated by reference in their entireties.

In exemplary embodiments, the locked nucleotides have a 2′ to 4′methylene bridge, as shown in structure A, for example. In otherembodiments, the bridge comprises a methylene or ethylene group, whichmay be substituted, and which may or may not have an ether linkage atthe 2′ position.

The oligonucleotide may comprise, consist essentially of, or consist of,an antisense sequence to a mRNA or miRNA. In one embodiment, theoligonucleotide comprises an antisense sequence directed to a miRNA. Forexample, the oligonucleotide comprises an antisense sequence that issufficiently complementary to a miRNA sequence to hybridize to theendogenous miRNA under physiological conditions. In such embodiments,the oligonucleotide can comprise a sequence that is at least partiallycomplementary to a mature miRNA sequence, e.g. at least about 75%, about80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%),or about 99% complementary to a mature miRNA sequence, such as, but notlimited to, a miRNA in Table 1, Table 2, or any one of the followingmiRNAs: miR-15a, miR-15h, miR-16-1, miR-16-2, miR-24, miR-25, miR-26a,miR-497, miR-195, miR-424, a let 7 family member, miR-21, miR-199a˜b,miR-214, miR-1 Oa-b, miR-16, miR-125b, miR-146a-b, miR-221, miR-222, amiR-30 family member, miR-126, miR-133, miR-1, miR-143, miR-145,miR-486, miR-92a, miR-320, miR-1-1, miR-1-2, miR-451, miR-378, miR-378*,miR-92, miR-34a, miR-34b, miR-34c, miR-29, or miR-33, In someembodiments, the miRNA is not miR208a, miR208b, or miR-499. In oneembodiment, the antisense oligonucleotide comprises a sequence that is100% complementary to a mature miRNA sequence, such as, but not limitedto, a miRNA selected from the group consisting of miR-15a, miR-15b,miR-16-1, miR-16-2, miR-24, miR-25, miR-26a, miR-497, miR-195, miR-424,a let 7 family member, miR-21, miR-199a-b, miR-214, miR-10a-b, miR-16,miR-125b, miR-146a-b, miR-221, miR-222, a miR-30 family member, miR-126,miR-133, miR-1, miR-143, miR-145, miR-486, miR-92a, miR-320, miR-1-1,miR-1-2, miR-451, miR-378, miR-378*, miR-92, miR-34a, miR-34b, miR-34c,miR-29, or miR-33. In some embodiments, the miRNA is not miR208a,miR208b, or miR-499. In some embodiments, the miRNA is not miR-208a,miR-208b, or miR-499.

The oligonucleotide generally has a nucleotide sequence designed totarget mature miRNA. The oligonucleotide may, in these or otherembodiments, also or alternatively be designed to target the pre- orpri-miRNA forms. In certain embodiments, the oligonucleotide may bedesigned to have a sequence containing from 1 to 5 (e.g., 1, 2, 3, or 4)mismatches relative to the fully complementary (mature) miRNA sequence.In some embodiments, the miRNA is not miR-208a, miR-208b, or miR-499. Incertain embodiments, such antisense sequences may be incorporated intoshRNAs or other RNA structures containing stem and loop portions, forexample.

In certain embodiments, the oligonucleotide comprises a nucleotidesequence that is completely complementary (i.e. fully complementary) toa nucleotide sequence of a miRNA. In some embodiments, the miRNA is notmiR-208a, miR-208b, or miR-499. In particular embodiments, theoligonucleotide comprises, consists essentially of, or consists of asequence completely complementary to the nucleotide sequence of a miRNA.In this context, “consists essentially of” includes the optionaladdition of nucleotides (e.g., one or two) on either or both of the 5′and 3′ ends, so long as the additional nucleotide(s) do notsubstantially affect (as defined by an increase in IC50 of no more than20%) the oligonucleotide's inhibition of the target miRNA activity inthe dual luciferase assay or mouse model.

The oligonucleotide can be from about 8 to about 20 nucleotides inlength, from about 18to about 50 nucleotides in length, from about 10 toabout 18 nucleotides in length, or from about 11 to about 16 nucleotidesin length. The oligonucleotide in some embodiments is about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, or about 18nucleotides in length. In some embodiments, theoligonucleotide is at least 16 nucleotides in length.

The oligonucleotide generally contains at least about 5, at least about7, or at least about 9 LNAs, but in various embodiments is not fullycomprised of LNAs. Generally, the number and position of LNAs is suchthat the oligonucleotide reduces mRNA or miRNA activity. In oneembodiment, the number and position of LNAs is such that theoligonucleotide has increased in vivo efficacy as compared to anoligonucleotide with a different number and/or position of LNAs. Incertain embodiments, the oligonucleotide does not contain a stretch ofnucleotides with more than four, or more than three, contiguous LNAs.For example, the oligonucleotide comprises no more than three contiguousLNAs. In these or other embodiments, the oligonucleotide can comprise aregion or sequence that is substantially or completely complementary toa miRNA seed region, in which the region or sequence comprises at leastthree, at least four, or at least five locked nucleotides. In someembodiments, the miRNA is not miR-208a, miR-208b, or miR-499,

In various embodiments, the oligonucleotide contains at least ninelocked nucleotides. For example, the oligonucleotide may contain ninelocked nucleotides and seven non-locked nucleotides. The pattern of LNAsmay be such that, from the 5′ end to the 3′ end of the oligonucleotide,at least positions 1, 6, 10, 13, and 15 are LNAs. In some embodiments,the pattern of LNAs may be such that, from the 5′ end to the 3′ end ofthe oligonucleotide, at least positions 1, 6, 10, 11, 13, and 16 areLNAs. In certain embodiments, from the 5′ end to the 3′ end of theoligonucleotide, positions 1, 5, 6, 8, 10, 11, 13, 15, and 16 are LNAs,and the remaining positions are non-locked nucleotides. In someembodiments, from the 5′ end to the 3′ end of the oligonucleotide,positions 1, 4, 5, 7, 9, 10, 12, 14, and 16 are LNAs, and remainingpositions are non-locked nucleotides. For example, in one embodiment, anoligonucleotide can comprise at least 16 nucleotides, in which from the5′ end to the 3′ end of the oligonucleotide, positions 1, 5, 6, 8, 10,11, 13, 15, and 16 are LNAs, and the remaining positions are non-lockednucleotides, wherein the oligonucleotide is a miRNA inhibitor.

For example, the oligonucleotide can comprise at least 16 nucleotides,in which from the 5′ end to the 3′ end of the oligonucleotide, positions1, 5, 6, 8, 10, 11, 13, 15, and 16 are LNAs, and the remaining positionsare non-locked nucleotides, the oligonucleotide is at least partiallycomplementary to a miRNA, in which the miRNA may in some embodiments,not be miR-208a, miR-208b, or miR-499. In another embodiment, theoligonucleotide can comprise at least 16nucleotides, in which from the5′ end to the 3′ end of the oligonucleotide, positions 1, 5, 6, 8, 10,11, 13, 14, and 16 are LNAs, and the remaining positions are non-lockednucleotides, the oligonucleotide is at least partially complementary toa miRNA, in which the miRNA may in some embodiments, not be miR-208a,miR-208b, or miR-499. In yet another example, the oligonucleotide cancomprise at least 16 nucleotides, in which from the 5′ end to the 3′ endof the oligonucleotide, positions 1, 5, 6, 8, 10, 11, 13, 15, and 16 areLNAs, and the remaining positions are non-locked nucleotides, theoligonucleotide is at least partially complementary to a seed region ofa miRNA, in which the miRNA may in some embodiments, not be miR-208a,miR-208b, or miR-499. In some embodiments, the oligonucleotide isselected from Tables 3, 5, 6, 7, 8, or 9. In certain embodiments, theoligonucleotide is a compound selected from M-10101, M-10707, M-11192,M-11185, M-10518, or M-11127.

For non-locked nucleotides, the nucleotide may contain a T modificationwith respect to a 2′ hydroxyl. For example, the 2′ modification may be2′ deoxy. Incorporation of 2′-modified. nucleotides in antisenseoligonucleotides may increase both resistance of the oligonucleotides tonucleases and their thermal stability with complementary RNA. Variousmodifications at the 2′ positions may be independently selected fromthose that provide increased nuclease sensitivity, without compromisingmolecular interactions with the RNA target or cellular machinery. Suchmodifications may be selected on the basis of their increased potency invitro or in vivo. Exemplary methods for determining increased potency(e.g., IC50) for miRNA inhibition are described herein, including thedual lueiferase assay and in vivo miRNA expression or targetde-repression.

In some embodiments the 2′ modification may be independently selectedfrom O-alkyl (which may be substituted), halo, and deoxy (H).Substantially all, or all, nucleotide 2′ positions of the non-lockednucleotides may be modified in certain embodiments, e.g., asindependently selected from O-alkyl (e.g., O-methyl), halo (e.g.,fluoro), deoxy (H), and amino. For example, the 2′ modifications mayeach be independently selected from O-methyl and fluoro. In exemplaryembodiments, purine nucleotides each have a 2′ OMe and pyrrolidinenucleotides each have a 2′-F. In certain embodiments, from one to aboutfive 2′ positions, or from about one to about three 2′ positions areleft unmodified (e.g., as 2′ hydroxyls).

2′ modifications in accordance with the invention also include smallhydrocarbon substituents. The hydrocarbon substituents include alkyl,alkenyl, alkynyl, and alkoxyalkyl, where the alkyl (including the alkylportion of alkoxy), alkenyl and alkynyl maybe substituted orunsubstituted. The alkyl, alkenyl, and alkynyl may be C1 to C10 alkyl,alkenyl or alkynyl, such as C1, C2, or C3. The hydrocarbon substituentsmay include one or two or three non-carbon atoms, which may beindependently selected from N, O, and/or S. The 2′ modifications mayfurther include the alkyl, alkenyl, and alkynyl as O-alkyl, O-alkenyl,and O-alkynyl.

Exemplary 2′ modifications in accordance with the invention include2′-O-alkyl (C1-3alkyl, such as 2′OMe or 2′OEt), 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethyiaminoethyioxyethyl (2′-O-DMAEOE), or 2′-O-N-methylacetamido(2′-O-NMA) substitutions.

In certain embodiments, the oligonucleotide contains at least one2′-halo modification (e.g., in place of a 2′ hydroxyl), such as2′-fluoro, 2′-chloro, 2′-bromo, and 2′-iodo. In some embodiments, the 2′halo modification is fluoro. The oligonucleotide may contain from 1 toabout 5 2′-halo modifications (e.g., fluoro), or from 1 to about 32′-halo modifications (e.g., fluoro). In some embodiments, theoligonucleotide contains all 2′-fluoro nucleotides at non-lockedpositions, or 2′-fluoro on all non-locked pyrimidine nucleotides. Incertain embodiments, the 2′-fluoro groups are independently di-, tri-,or un-methylated.

The oligonucleotide may have one or more 2′-deoxy modifications (e.g., Hfor 2′ hydroxyl), and in some embodiments, contains from 2 to about 102′-deoxy modifications at non-locked positions, or contains 2′deoxy atail non-locked positions.

In exemplary embodiments, the oligonucleotide contains 2′ positionsmodified as 2′OMe in non-locked positions. Alternatively, non-lockedpurine nucleotides are modified at the 2′ position as 2′OMe, withnon-locked pyrimidine nucleotides modified at the 2′ position as2′-fluoro.

In certain embodiments, the oligonucleotide further comprises at leastone terminal modification or “cap”. The cap may be a 5′ and/or a 3′-capstructure. The terms “cap” or “end-cap” include chemical modificationsat either terminus of the oligonucleotide (with respect to terminalribonucleotides), and including modifications at the linkage between thelast two nucleotides on the 5′ end and the last two nucleotides on the3′ end. The cap structure as described herein may increase resistance ofthe oligonucleotide to exonucleases without compromising molecularinteractions with the RNA target or cellular machinery. Suchmodifications may be selected on the basis of their increased potency invitro or in vivo. The cap can be present at the 5′-terminus (5′-cap) orat the 3′-terminus (3′-cap) or can be present on both ends. In certainembodiments, the 5′- and/or 3′-cap is independently selected fromphosphorothioate monophosphate, abasic residue (moiety),phosphorothioate linkage, 4′-thio nucleotide, carbocyclic nucleotide,phosphorodithioate linkage, inverted nucleotide or inverted abasicmoiety (2′-3′ or 3′-3′), phosphorodithioate monophosphate, andmethylphosphonate moiety. The phosphorothioate or phosphorodithioatelinkage(s), when part of a cap structure, are generally positionedbetween the two terminal nucleotides on the 5′ end and the two terminalnucleotides on the 3′ end.

In certain embodiments, the oligonucleotide has at least one terminalphosphorothioate monophosphate. The phosphorothioate monophosphate maysupport a higher potency by inhibiting the action of exonucleases. Thephosphorothioate monophosphate may be at the 5′ and/or 3′ end of theoligonucleotide. A phosphorothioate monophosphate is defined by thefollowing structures, where B is base, and R is a 2′ modification asdescribed above:

Where the cap structure can support the chemistry of a lockednucleotide, the cap structure may incorporate a LNA as described herein.

Phosphorothioate linkages may be present in some embodiments, such asbetween the last two nucleotides on the 5′ and the 3′ end (e.g., as partof a cap structure), or as alternating with phosphodiester bonds. Inthese or other embodiments, the oligonucleotide may contain at least oneterminal abasic residue at either or both the 5′ and 3′ ends. An abasicmoiety does not contain a commonly recognized purine or pyrimidinenucleotide base, such as adenosine, guanine, cytosine, uracil orthymine. Thus, such abasic moieties lack a nucleotide base or have othernon-nucleotide base chemical groups at the 1′ position. For example, theabasic nucleotide maybe a reverse abasic nucleotide, e.g., where areverse abasic phosphoramidite is coupled via a 5′ amidite (instead of3′ amidite) resulting in a 5′-5′ phosphate bond. The structure of areverse abasic nucleoside for the 5′ and the 3′ end of a polynucleotideis shown below.

The oligonucleotide may contain one or more phosphorothioate linkages.Phosphorothioate linkages have been used to render oligonucleotides moreresistant to nuclease cleavage. For example, the polynucleotide may bepartially phosphorothioate-linked, for example, phosphorothioatelinkages may alternate with phophodiester linkages. In certainembodiments, however, the oligonucleotide is fullyphosphorothioate-linked. In other embodiments, the oligonucleotide hasfrom one to five or one to three phosphate linkages.

In some embodiments, the nucleotide has one or more carboxamido-modifiedbases as described in WO 2012/061810, which is hereby incorporated byreference, including with respect to all exemplary pyrimidinecarboxamido modifications disclosed therein with heterocyclicsubstituents.

The synthesis of oligonucleotides, including modified polynucleotides,by solid phase synthesis is well known and is reviewed in New ChemicalMethods for Synthesizing Polynucleotides. Caruthers M H, Beaucage S L,Efcavitch J W, Fisher E F, Matteucci M D, Stabinsky Y. Nucleic AcidsSymp. Ser. 1980; (7):215-23.

The oligonucleotide may be incorporated within a variety ofmacromolecular assemblies or compositions. Such complexes for deliverymay include a variety of liposomes, nanoparticles, and micelles,formulated for delivery to a patient. The complexes may include one ormore fusogenic or lipophilic molecules to initiate cellular membranepenetration. Such molecules are described, for example, in U.S. Pat. No.7,404,969 and U.S. Pat. No. 7,202,227, which are hereby incorporated byreference in their entireties. Alternatively, the oligonucelotide mayfurther comprise a pendent lipophilic group to aid cellular delivery,such as fatty acids and those described in WO 2010/129672, which ishereby incorporated by reference in its entirety. In some embodiments,the oligonucleotide may further comprise a pendent hydrophilic group totarget the oligonucleotide to particular tissues. For instance, in oneembodiment, the oligonucleotide may be conjugated to a sugar moiety,such as mannose-6-phosphate or an amino sugar, such as N-acetylglucosamine.

The oligonucleotides of the invention may be formulated as a variety ofpharmaceutical compositions. Pharmaceutical compositions will beprepared in a form appropriate for the intended application. Generally,this will entail preparing compositions that are essentially free ofpyrogens, as well as other impurities that could be harmful to humans oranimals. Exemplary delivery/formulation systems include colloidaldispersion systems, macromolecule complexes, nanocapsules,nanoparticles, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.Commercially available fat emulsions that are suitable for deliveringthe nucleic acids of the invention to cardiac and skeletal muscletissues include Intralipid®, Liposyn®, Liposyn® II, Liposyn® III,Nutrilipid, and other similar lipid emulsions. A preferred colloidalsystem for use as a delivery vehicle in vivo is a liposome (i.e., anartificial membrane vesicle). The preparation and use of such systems iswell known in the art. Exemplary formulations are also disclosed in U.S.Pat. Nos. 5,981,505; 6,217,900 6,383,512; 5,783,565; 7,202,227;6,379,965; 6,127,170; 5,837,533; 6,747,014; and WO03/093449, all ofwhich are hereby incorporated by reference in their entireties.

The compositions or formulations may employ a plurality of therapeuticoligonucleotides, including at least one described herein. For example,the composition or formulation may employ at least 2, 3, 4, or 5 miRNAinhibitors described herein. In another embodiment, an oligonucleotideof the present invention may be used in combination with othertherapeutic modalities. Combinations may also be achieved by contactingthe cell with more than one distinct compositions or formulations, atthe same time. Alternatively, combinations may be administeredsequentially.

In some embodiments, the oligonucleotide is formulated for conventionalsubcutaneous or intravenous administration, for example, by formulatingwith appropriate aqueous diluent, including sterile water and normalsaline.

The pharmaceutical compositions and formulations may employ appropriatesalts and buffers to render deliver vehicles stable and allow for uptakeby target cells. Aqueous compositions of the present invention comprisean effective amount of the delivery vehicle comprising the inhibitoroligonucleotide (e.g. liposomes, nanoparticles, or other complexes),dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium. The phrases “pharmaceutically acceptable” or“pharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” may include one or more solvents,buffers, solutions, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the likeacceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Supplementary active ingredients also can be incorporated intothe compositions.

Administration or delivery of the pharmaceutical compositions accordingto the present invention may be via any route so long as the targettissue is available via that route. For example, administration may betopical or by intradermal, subcutaneous, intramuscular, intraperitoneal,intraarterial, intracoronary, intrathecal, or intravenous injection, orby direct injection into target tissue (e.g., cardiac tissue). Thestability and/or potency of the oligonucleotides disclosed herein allowsfor convenient routes of administration, including subcutaneous,intradermal, intravenous, and intramuscular. Pharmaceutical compositionscomprising an oligonucleotide described herein may also be administeredby catheter systems or systems that isolate coronary circulation fordelivering therapeutic agents to the heart. Various catheter systems fordelivering therapeutic agents to the heart and coronary vasculature areknown in the art. Some non-limiting examples of catheter-based deliverymethods or coronary isolation methods suitable for use in the presentinvention are disclosed in U.S. Pat. Nos. 6,416,510; 6,716,196; and6,953,466; PC T Publication Nos. WO 2005/082440 and WO 2006/089340; andU.S. Patent Publication Nos. 2007/0203445, 2006/0148742, and2007/0060907, which are all hereby incorporated by reference in theirentireties.

The compositions or formulations may also be administered parenterallyor intraperitoneally. By way of illustration, solutions of theconjugates as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations generallycontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use or catheterdelivery include, for example, sterile aqueous solutions or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersions. Generally, these preparations aresterile and fluid to the extent that easy injectability exists.Preparations should be stable under the conditions of manufacture andstorage and should be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Appropriate solvents ordispersion media may contain, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetable oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialan antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating theconjugates in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the desired other ingredients, e.g., as enumerated above. Inthe case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation include vacuum-dryingand freeze-drying techniques which yield a powder of the activeingredient(s) plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drag releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologies standards.

The present invention provides a method for delivering oligonucleotidesto a cell (e.g., as part of a composition or formulation describedherein), and methods for treating, ameliorating, or preventing theprogression of a condition in a subject. As used herein, the term“subject” or “patient” refers to any vertebrate including, withoutlimitation, humans and other primates (e.g., chimpanzees and other apesand monkey species), farm animals (e.g., cattle, sheep, pigs, goats andhorses), domestic mammals (e.g., dogs and cats), laboratory animals(e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g.,domestic, wild and game birds such as chickens, turkeys and othergallinaceous birds, ducks, geese, and the like). In some embodiments,the subject is a mammal. In other embodiments, the subject is a human.

The oligonucleotide or pharmaceutical composition may be contacted invitro or in vivo with a target cell (e.g., a mammalian cell). The cellmay be a kidney, liver, vascular, or heart cell.

The method generally comprises administering the oligonucleotide orcomposition comprising the same to a subject or cell. Theoligonucleotide, as described herein, can be mRNA or miRNA inhibitor. Insome embodiments, the miRNA inhibitor is not a miR-208a inhibitor,miR-208b inhibitor, or miR-499 inhibitor. Thus, the patient may have acondition associated with, mediated by, or resulting from, expression ordysregulation of a mRNA or miRNA. Such conditions include, but are notlimited to, cardiovascular conditions, such as cardiac hypertrophy,myocardial infarction, heart failure (e.g., congestive heart failure),myocardial ischemia, ischemia-reperfusion injury, vascular damage,coronary artery disease, peripheral artery disease, vulnerable plaque,restenosis, or pathologic cardiac fibrosis. Other conditions may includemetabolic conditions, renal conditions (e.g., renal ischemia), hepaticconditions, or pulmonary conditions. Thus, the invention provides a useof the modified oligonucleotides and compositions of the presentinvention for treating such conditions, and for the preparation ofmedicaments for such treatments.

In certain embodiments, the subject (e.g., human patient) has one ormore risk factors for a condition, such as, but not limited to, longstanding uncontrolled hypertension, uncorrected valvular disease,chronic angina, recent myocardial infarction, congestive heart failure,congenital predisposition to heart disease and pathological hypertrophy.Alternatively or in addition, the patient may have been diagnosed ashaving a genetic predisposition to, for example, cardiac hypertrophy, ormay have a familial history of, for example, cardiac hypertrophy.

In this aspect, the present invention may provide for an improvedexercise tolerance, reduced hospitalization, better quality of life,decreased morbidity, and/or decreased mortality in a patient with heartfailure or cardiac hypertrophy.

In certain embodiments, the activity of the miRNA in a tissue ofinterest, such as cardiac tissue, or as determined in serum, is reducedor inhibited.

In various embodiments, the pharmaceutical composition is administeredby parenteral administration or by direct injection into heart tissue.The parenteral administration may be intravenous, subcutaneous, orintramuscular. In some embodiments, the composition is administered byoral, transdermal, sustained release, controlled release, delayedrelease, suppository, catheter, or sublingual administration. In certainembodiments, the oligonucleotide is administered at a dose of about 25mg/kg or less, or a dose of about 10 mg/kg or less, or a dose of about 5mg/kg or less. In these embodiments, the oligonucleotide or compositionmay be administered by intramuscular or subcutaneous injection, orintravenously.

In some embodiments, the methods further comprise scavenging or clearingthe miRNA inhibitors following treatment. For example, a polynucleotidehaving a nucleotide sequence that is complementary to the inhibitor(e.g., a polynucleotide comprising a miRNA sequence) may be administeredafter therapy to attenuate or stop the function of the inhibitor.

The present invention is further illustrated by the following additionalexamples that should not be construed as limiting. Those of skill in theart should, in light of the present disclosure, appreciate that manychanges can be made to the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

All publications and patents and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

EXAMPLES Example 1 In vivo Efficacy of AntimiR-208a

To determine whether the location of the LNA base affects in vivoefficacy of microRNA inhibitors (antimiRs) of identical length and LNApercentage, several antimiRs with different LNA modification patternswere designed and tested for efficacy in inhibiting miRNA function invivo.

Sixteen antimiRs against miR-208a (FIG. 1A) with varying Tm measurementswere designed, as depicted in Table 3 below:

TABLE 3 LNA/ Predicted Molecule # Alias Sequence Length DNA Tm M-10101208a_LNA_DNA_16_PS lCs; dTs; dTs; dTs; lTs; lTs; dGs; lCs; dTs; lCs;lGs; dTs; lCs; dTs; lTs; lA 16 9/7 81 (SEQ ID NO: 76) M-10679 208a LNAlCs; dTs; lTs; dTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; lTs; dCs; lTs;lTs; dA 16 9/7 93 C_T_DNA_16_1 (SEQ ID NO: 77) M-10680 208a_LNA_opt_1lCs; dTs; lTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs; dTs; lCs; dTs;dTs; lA 16 9/7 90 (SEQ ID NO: 78) M-10681 208a_LNA_opt_2 lCs; dTs; lTs;lTs; dTs; lTs; dGs; lCs; lTs; lCs; dGs; dTs; lCs; dTs; lTs; dA 16 9/7 93(SEQ ID NO: 79) M-10682 208a_LNA_opt_3 lCs; dTs; lTs; dTs; lTs; dTs;lGs; dCs; lTs; dCs; lGs; dTs; lCs; dTs; lTs; lA 16 9/7 86 (SEQ ID NO:80) M-10683 208a_LNA_opt_4 lCs; dTs; dTs; lTs; lTs; dTs; lGs; dCs; lTs;lCs; dGs; lTs; dCs; lTs; dTs; lA 16 9/7 92 (SEQ ID NO: 81) M-10673208a_LNA_opt_5 lCs; dTs; lTs; lTs; lTs; lTs; dGs; lCs; dTs; lCs; dGs;dTs; lCs; dTs; lTs; dA 16 9/7 93 (SEQ ID NO: 82) M-11184 208a_10626 lCs;dTs; lTs; lTs; dTs; dTs; lGs; lCs; dTs; lCs; dGs; lTs; dCs; lTs; dTs; lA16 9/7 83 (SEQ ID NO: 83) M-11293 208a_scr2_1 lCs; dTs; dTs; dTs; lTs;lTs; dGs; dCs; lTs; lCs; lGs; dTs; lCs; dTs; lTs; lA 16 9/7 86 (SEQ IDNO: 84) M-11294 208a_scr2_2 lCs; lTs; dTs; dTs; dTs; lTs; dGs; lCs; dTs;lCs; lGs; dTs; lCs; dTs; lTs; lA 16 9/7 77 (SEQ ID NO: 85) M-11295208a_scr2_3 lCs; lTs; dTs; dTs; dTs; lTs; lGs; dCs; dTs; lCs; lGs; lTs;dCs; lTs; dTs; lA 16 9/7 76 (SEQ ID NO: 86) M-11296 208a_scr2_4 lCs;dTs; dTs; dTs; lTs; lTs; dGs; lCs; dTs; lCs; lGs; dTs; lCs; lTs; dTs; lA16 9/7 80 (SEQ ID NO: 87) M-11297 208a_scr2_5 lCs; lTs; dTs; dTs; lTs;dTs; dGs; lCs; lTs; lCs; dGs; lTs; dCs; lTs; dTs; lA 16 9/7 87 (SEQ IDNO: 88) M-11298 208a_scr2_6 lCs; dTs; lTs; dTs; lTs; dTs; lGs; lCs; dTs;lCs; dGs; lTs; dCs; lTs; dTs; lA 16 9/7 83 (SEQ ID NO: 89) M-11299208a_scr2_7 lCs; dTs; dTs; lTs; lTs; dTs; lGs; lCs; dTs; lCs; dGs; lTs;dCs; lTs; dTs; lA 16 9/7 83 (SEQ ID NO: 90) M-11300 208a_scr2_8 lCs;lTs; dTs; dTs; lTs; dTs; lGs; dCs; lTs; lCs; dGs; lTs; dCs; lTs; dTs; lA16 9/7 88 (SEQ ID NO: 91)

TABLE 4 Description of Notations deoxy A dA deoxy G dG deoxy C dC deoxyT dT lna A lA lnaG lG lna C lC lna T lT deoxy A P = S dAs deoxy G P = SdGs deoxy C P = S dCs deoxy T P = S dTs lna A P = S lAs lnaG P = S lGslna C P = S lCs lna T P = S lTs

These antimiRs were dorsally injected into 6-8 week old Sprague Dawieyrats subcutaneously at a dose of 25 mg/kg (n=4 per group). Injectionvolume was 1.0 mL. A control oligonucleotide with similar LNA and DNApercentage (9/7) was also used as a chemistry control. This moleculenumber is M-10591 and was designed to target a C. elegans—specificmiRNA. Four days after a single dose, these rats were sacrificed andplasma was collected for liver and kidney toxicology parameters.Additionally, heart, liver, and kidney were collected for molecularanalysis including miRNA inhibition, target de-repression, andantimiR-distribution quantification. RNA was isolated from cardiactissue and real-time PCR was performed. All antimiRs designed againstmiR-208a showed significant inhibition of miR-208a suggesting ailantimiRs were delivered to cardiac tissue (FIG. 1B). To determine ifmiR-208a inhibition correlated to in vivo efficacy, miR˜208a targetswere assessed for de-repression by performing real-time PGR for themiR-208a target, Dynlt1. Surprisingly, only four of the sixteen antimiRstested showed significant de-repression of Dynlt1 (FIG. 1C).

To determine if treatment with any of these antimiRs resulted inelevated liver and/or kidney toxicology parameters, ELISAs for ALT, AST,and BUN were performed to assess liver and kidney function. NoantimiR-treated group showed any elevation in either liver or kidneytoxicology parameters (FIG. 1D).

To determine if the difference in efficacy between compounds is due tobetter cardiac distribution for the efficacious molecules, antimiRdistribution to the heart, liver, and kidney for 2 antimiRs that showedefficacy (M-10101 and M-10683) and 2 antimiRs that did not show efficacy(M-10673 and M-10681) were assessed. ELISA-based distribution analysesshowed no better cardiac presence for the efficacious compounds comparedto the non-efficacious compounds. In fact, the non-efficacious compoundsappeared to show better distribtuion to all tissues. (FIG. 1E).

These data suggest different LNA and DNA placement within the antimiR,results in. significantly different antimiR efficacy as it pertains tothe heart, with the LNA/DNA “motif” of M-10101 sequence appearing to bethe best compound for cardiac efficacy.

Example 2 In vivo Efficacy of AntimiR-208b

To test if the efficacious LNA/DNA motif of M-10101 remains efficaciousfor additional miRNAs, a subset of these for other miRNAs, includingmiR-208b, miR-29, miR-378, miR-199a, and miR-92a was tested. Allexperimental designs were the same as performed for miR-208a asdescribed in Example 1.

Nine antimiRs against miR-208b with LNA and DNA placements similar tothose found for the miR-208a screen were synthesized (FIG. 2A), withvarying Tm measurements was designed, as depicted in Table 5 below(description of notations is as described in Table 4):

TABLE 5 LNA/ Predicted Molecule # Alias Sequence Length DNA Tm M-10707208b_10101 lCs; dCs; dTs; dTs; lTs; lTs; dGs; lTs; dTs; lCs; lGs; dTs;lCs; dTs; lTs; lA 16 9/7 82 (SEQ ID NO: 92) M-11283 208b_10679 lCs; dCs;lTs; dTs; lTs; lTs; dGs; lTs; dTs; lCs; dGs; lTs; dCs; lTs; lTs; dA 169/7 91 (SEQ ID NO: 93) M-11284 208b_10680 lCs; dCs; lTs; lTs; lTs; lTs;dGs; lTs; dTs; lCs; dGs; dTs; lCs; dTs; dTs; lA 16 9/7 89 (SEQ ID NO:94) M-11285 208b_10681 lCs; dCs; lTs; lTs; dTs; lTs; dGs; lTs; lTs; lCs;dGs; dTs; lCs; dTs; lTs; dA 16 9/7 94 (SEQ ID NO: 95) M-11286 208b_10682lCs; dCs; lTs; dTs; lTs; dTs; lGs; dTs; lTs; dCs; lGs; dTs; lCs; dTs;lTs; lA 16 9/7 86 (SEQ ID NO: 96) M-11287 208b_10683 lCs; dCs; dTs; lTs;lTs; dTs; lGs; dTs; lTs; lCs; dGs; lTs; dCs; lTs; dTs; lA 16 9/7 93 (SEQID NO: 97) M-11288 208b_10673 lCs; dCs; lTs; lTs; lTs; lTs; dGs; lTs;dTs; lCs; dGs; dTs; lCs; dTs; lTs; dA 16 9/7 91 (SEQ ID NO: 98) M-11289208b_10626 lCs; dCs; lTs; lTs; dTs; dTs; lGs; lTs; dTs; lCs; dGs; lTs;dCs; lTs; dTs; lA 16 9/7 89 (SEQ ID NO: 99) M-11290 208b_LNA_opt6 lCs;lCs; dTs; dTs; lTs; dTs; lGs; lTs; dTs; lCs; dGs; lTs; dCs; lTs; dTs; lA16 9/7 92 (SEQ ID NO: 100)

These antimiRs were dorsally injected into 6-8 week old Sprague Dawleyrats subcutaneously at a dose of 25 mg kg (n=4 per group). Injectionvolume was 1.0 ml. A control oligonucleotide with similar LNA and DNApercentage (9/7) was also used as a chemistry control. This moleculenumber is M-10591 and was designed to target a C. elegans—specificmiRNA. Four days after a single dose, these rats were sacrificed and theheart was collected for molecular analysis including miRNA inhibitionand target de-repression. RNA was isolated from cardiac tissue andreal-time PGR was performed. All antimiRs designed against miR-208bshowed significant inhibition of miR-208b suggesting all antimiRs weredelivered to cardiac tissue (FIG. 2B). To determine if miR-208binhibition correlated to in vivo efficacy, the miR-208b target, Dynlt1,was assessed for de-repression by performing real-time PGR.Surprisingly, only M-10707 showed significant de-repression of Dynlt1(FIG. 2C), which is the same LNA/DNA motif that showed the best efficacyfor miR-208a. (FIG. 1C).

These data suggest the LNA/DNA motif of M-l 0101 and M-10707 (which isthe same) confers cardiac efficacy in vivo.

Example 3 In vivo Efficacy of AntimiR-378

To determine if motif M-10101 extends beyond the miR-208 family, 7antimiRs against miR-378 with LNA and DNA placements similar to thosefound for the miR-208a screen (FIG. 3A), with varying Tin measurementsas depicted in Table 6 below (description of notations is as describedin Table 4), were designed and synthesized:

TABLE 6 LNA/ Predicted Molecule # Alias Sequence Length DNA Tm M-11192378_10101 lCs; dTs; dGs; dAs; lCs; lTs; dCs; lCs; dAs; lAs; lGs; dTs;lCs; dCs; lAs; lGs 16 9/7 86 (SEQ ID NO: 101) M-11193 378_10680 lCs;dTs; lGs; lAs; lCs; lTs; dCs; lCs; dAs; lAs; dGs; dTs; lCs; dCs; dAs;lGs 16 9/7 89 (SEQ ID NO: 102) M-11194 378_10681 lCs; dTs; lGs; lAs;dCs; lTs; dCs; lCs; lAs; lAs; dGs; dTs; lCs; dCs; lAs; dGs 16 9/7 89(SEQ ID NO: 103) M-11195 378_10682 lCs; dTs; lGs; dAs; lCs; dTs; lCs;dCs; lAs; dAs; lGs; dTs; lCs; dCs; lAs; lGs 16 9/7 86 (SEQ ID NO: 104)M-11196 378_10683 lCs; dTs; dGs; lAs; lCs; dTs; lCs; dCs; lAs; lAs; dGs;lTs; dCs; lCs; dAs; lGs 16 9/7 91 (SEQ ID NO: 105) M-11197 378_10673lCs; dTs; lGs; lAs; lCs; lTs; dCs; lCs; dAs; lAs; dGs; dTs; lCs; dCs;lAs; dGs 16 9/7 95 (SEQ ID NO: 106) M-11198 378_10626 lCs; dTs; lGs;lAs; dCs; dTs; lCs; lCs; dAs; lAs; dGs; lTs; dCs; lCs; dAs; lGs 16 9/795 (SEQ ID NO: 107)

These antimiRs were dorsally injected into 6-8 week old Sprague Dawleyrats subcutaneously at a dose of 25 mg/kg (n=4 per group). Injectionvolume was 1.0 ml. A control oligonucleotide with similar LNA, and DNApercentage (9/7) was also used as a chemistry control. This moleculenumber is M-10591 and was designed to target a C. elegans—specificmiRNA. Four days after a single dose, these rats were sacrificed and theheart was collected for molecular analysis including miRNA inhibitionand target de-repression. RNA was isolated from cardiac tissue andreal-time PCR was performed. All antimiRs designed against miR-378showedsignificant inhibition of miR-378 suggesting all antimiRs were deliveredto cardiac tissue (FIG. 3B). To determine if miR-378 inhibitioncorrelated to in vivo efficacy, we assessed the miR-378 target, Gfpt2,for de-repression by performing real-time PCR. Surprisingly, onlyM-11192 showed significant de-repression of Gfpt2 (FIG. 3C), which isthe same LNA/DNA motif that showed the best efficacy for miR-208a andmiR-208b in the heart. (FIGS. 1C and 2C).

These data highly suggest the LNA/DNA motif of M-10101, M-10707, andM-11192(which is the same) confers cardiac efficacy in vivo.

Example 4 In vivo Efficacy of AntimiR-29

Seven antimiRs against miR-29b with LNA and DNA placements similar tothose found for the miR-208a screen (FIG. 4A) were synthesized todetermine if this motif confers efficacy in further miRNA families. Thesequence and modification patterns of these antimiRs with theircorresponding predicted Tm values are depicted in Table 7 below(description of notations is as described in Table 4):

TABLE 7 LNA/ Predicted Molecule # Alias Sequence Length DNA Tm 1118529b_10101 lGs; dAs; dTs; dTs; lTs; lCs; dAs; lAs; dAs; lTs; lGs; dGs;lTs; dGs; lCs; lTs 16 9/7 84 (SEQ ID NO: 108) 11186 29b_10680 lGs; dAs;lTs; lTs; lTs; lCs; dAs; lAs; dAs; lTs; dGs; dGs; lTs; dGs; dCs; lTs 169/7 91 (SEQ ID NO: 109) 11187 29b_10681 lGs; dAs; lTs; lTs; dTs; lCs;dAs; lAs; lAs; lTs; dGs; dGs; lTs; dGs; lCs; dTs 16 9/7 87 (SEQ ID NO:110) 11188 29b_10682 lGs; dAs; lTs; dTs; lTs; dCs; lAs; dAs; lAs; dTs;lGs; dGs; lTs; dGs; lCs; lTs 16 9/7 82 (SEQ ID NO: 111) 11189 29b_10683lGs; dAs; dTs; lTs; lTs; dCs; lAs; dAs; lAs; lTs; dGs; lGs; dTs; lGs;dCs; lTs 16 9/7 85 (SEQ ID NO: 112) 11190 29b_10673 lGs; dAs; lTs; lTs;lTs; lCs; dAs; lAs; dAs; lTs; dGs; dGs; lTs; dGs; lCs; dTs 16 9/7 96(SEQ ID NO: 113) 11191 29b_10626 lGs; dAs; lTs; lTs; dTs; dCs; lAs; lAs;dAs; lTs; dGs; lGs; dTs; lGs; dCs; lTs 16 9/7 82 (SEQ ID NO: 114)

These antimiRs were dorsally injected into 6-8 week old Sprague Dawleyrats subcutaneously at a dose of 25 mg/kg (n=4 per group). Injectionvolume was 1.0 mL. A control oligonucleotide with similar LNA and DNApercentage (9/7) was also used as a chemistry control. This moleculenumber is M-10591 and was designed to target a C. elegans—specificmiRNA. Four days after a single dose, these rats were sacrificed and theheart, liver, and kidney were collected for molecular analysis includingmiRNA inhibition, target de-repression, and antimiR-distributionquantification, RNA was isolated from cardiac, hepatic, and renal tissueand real-time PCR was performed. All antimiRs designed against miR-29showed significant inhibition of miR-29 family members in all tissuessuggesting all antimiRs were delivered to these three tissues (FIG. 4B).

To determine if miR-29 family inhibition correlated with in vivoefficacy, the miR-29targets, Mcll and DnmtSb, were assess forde-repression by performing real-time PGR. Surprisingly, only M-11185showed significant de-repression of Mcll in the heart and trendingde-repression of DnmtSh (FIG. 4C), which is the same LNA/DNA motif thatshowed the best efficacy for miR-208a, miR-208b, and miR-378 in theheart. (FIGS. 1C, 2C, and 3C). Surprisingly, all antimiR-29 compoundsappeared to show de-repression of Mcll in the liver, furthering thenotion that this motif confers cardiac efficacy while the othercompounds are active in other tissues.

To determine if the difference in efficacy between compounds is due tobetter cardiac distribution for the efficacious molecules, we quantifiedantimiR distribution to the heart and liver for all antimiR-29compounds. ELISA-based distribution analyses showed no better cardiacpresence for the most efficacious compound (M-11185) compared to theless efficacious compounds. For hepatic tissue where efficacy wassimilar among compounds, distribution was similar as well (FIG. 4D).

Example 5 In vivo Efficacy of AntimiR-199

Five antimiRs against miR-199a with LNA and DNA placements similar tothose found for the miR-208a screen (FIG. 5A) were synthesized todetermine if this motif confers efficacy in further miRNA families. Thesequence and modification patterns of these antimiRs with theircorresponding predicted Tm values are depicted in Table 8 below(description of notations is as described in Table 4). The M-l0518compound contains the same LNA and DNA placements as M-10101(antimiR-208a), M-10707 (antimiR-208b), M-l 1192 (antimiR-378), andM-11185 (antimiR-29).

TABLE 8 LNA/ Predicted Molecule # Alias Sequence Length DNA Tm 10518199a_10101 lGs; dTs; dAs; dGs; lTs; lCs; dTs; lGs; dAs; lAs; lCs; dAs;lCs; dTs; lGs; lGs 16 9/7 93 (SEQ ID NO: 115) 11390 199a_10293 lGs; dTs;dAs; dGs; lTs; lCs; dTs; dGs; lAs; lAs; lCs; dAs; lCs; dTs; lGs; lGs 169/7 92 (SEQ ID NO: 116) 11391 199a_10294 lGs; lTs; dAs; dGs; dTs; lCs;dTs; lGs; dAs; lAs; lCs; dAs; lCs; dTs; lGs; lGs 16 9/7 86 (SEQ ID NO:117) 11392 199a_10296 lGs; dTs; dAs; dGs; lTs; lCs; dTs; lGs; dAs; lAs;lCs; dAs; lCs; lTs; dGs; lGs 16 9/7 92 (SEQ ID NO: 118) 11393 199a_10683lGs; dTs; dAs; lGs; lTs; dCs; lTs; dGs; lAs; lAs; dCs; lAs; dCs; lTs;dGs; lGs 16 9/7 86 (SEQ ID NO: 119)

These antimiRs were dorsally injected into 6-8 week old Sprague Dawleyrats subcutaneously at a dose of 25 mg/kg (n=4 per group). Injectionvolume was 1.0 raL. A control oligo with similar LNA and DNA percentage(9/7) was also used as a chemistry control. This molecule number isM-l0591 and was designed to target a C. elegans—specific miRNA. Fourdays after a single dose, these rats were sacrificed and plasma wascollected for liver and kidney toxicology parameters. Additionally,heart, lung, liver, and kidney were collected for molecular analysisincluding miRNA inhibition and target de-repression. RNA was isolatedfrom cardiac, pulmonary, hepatic, and renal tissue and real-time PCR wasperformed. All antimiRs designed against miR-199a showed significantinhibition of miR-199a in all tissues suggesting all antimiRs weredelivered to these four tissues (FIG. 5B).

To determine if miR-199a inhibition correlated to in vivo efficacy, weassessed the miR-199 target, Ddrl for de-repression by performingreal-time PCR. Surprisingly, all antimiRs targeting miR-199a appeared toshow Ddrl target de-repression in the heart with the exception ofM-11390 (FIG. 5C). For other tissues, different compounds showed varyingdegrees of target regulation, however, M-10518 (which is the M-10101motif) consistently appeared to show target de-repression for alltissues, suggesting this motif confers cardiac and multi-tissue efficacyin vivo.

Example 6 In vivo Efficacy of AntimiR-92a

Three antimiRs against miR-92a with LNA and DNA placements similar tothose found for the miR-208a screen were synthesized to determine ifthis motif confers efficacy in further miRNA families. The sequence andmodification patterns of these antimiRs with their correspondingpredicted Tm values are depicted in Table 9 below (description ofnotations is as described in Table 4). The M-11127 compound contains thesame LNA and DNA placements as M-10101 (antimiR-208a), M-10707(antimiR-208b), M-11192 (antimiR-378), M-11185(antimiR-29), and M-10518(antimiR-199a).

TABLE 9 Molecule LNA/ Predicted # Alias Sequence Length DNA Tm 1033892a_LNA_16_PS lCs; dCs; lGs; dGs; dGs; lAs; dCs; lAs; dAs; lGs; lTs;dGs; lCs; lAs; dAs; lT 16 9/7 85 (SEQ ID NO: 120) 11127 92a_LNA_16 _1lCs; dCs; dGs; dGs; lGs; lAs; dCs; lAs; dAs; lGs; lTs; dGs; lCs; dAs;lAs; lT 16 9/7 89 (SEQ ID NO: 121) 11130 92a_LNA_16_4 lCs; dCs; lGs;dGs; dGs; lAs; dCs; dAs; lAs; lGs; lTs; dGs; lCs; lAs; dAs; lT 16 9/7 86(SEQ ID NO: 122)

These antimiRs were dorsally injected into 6-8 week old Sprague Dawleyrats subcutaneously at a dose of 25 mg/kg (n=4 per group). Injectionvolume was 1.0 mL. Two days after a single dose, these rats weresacrificed and endothelial cells from the heart were collected formolecular analysis including miRNA inhibition and target de-repression.RNA was isolated from endothelial cells and real-time PGR was performedto assess de-repression of the miR-92a target, Map2K4. Administration ofantimiR M-11127 (which is the M-10101 motif) as well as antimiR M-11130resulted in a significant increase in Map2K4 expression in endothelialcells (FIG. 6), demonstrating that these two inhibitors have in vivoefficacy.

1. An oligonucleotide comprising a sequence complementary to the seedregion of a miRNA, wherein the sequence comprises at least five lockednucleic acids (LNAs) and the oligonucleotide comprises at least onenon-locked nucleotide, and the oligonucleotide has increased in vivoefficacy as compared to a second oligonucleotide comprising the samesequence and LNA composition and different LNA motif.
 2. Theoligonucleotide of claim 1, wherein the oligonucleotide comprises a LNAat the 5′ end and/or 3′ end.
 3. (canceled)
 4. The oligonucleotide ofclaim 1, wherein the oligonucleotide comprises no more than threecontiguous LNAs.
 5. The oligonucleotide of claim 1, wherein theoligonucleotide is at least 16 nucleotides in length.
 6. Anoligonucleotide comprising a sequence of 16 nucleotides, wherein thesequence comprises at least five locked nucleic acids (LNAs), a LNA atthe 5′ end, a LNA at the 3′ end, and no more than three contiguous LNAs.7. The oligonucleotide of claim 6, wherein from the 5′ end to the 3′end, positions 1, 5, 6, 8, 10, 11, 13, 15, and 16 of the sequence areLNAs.
 8. The oligonucleotide of claim 1, wherein at least one of thenon-locked nucleotides is 2′ deoxy, 2′ O-alkyl, or T halo.
 9. Theoligonucleotide of claim 1, wherein all of the non-locked nucleotidesare 2′ deoxy.
 10. (canceled)
 11. The oligonucleotide of claim 1, whereinat least one LNA has a 2′ to 4′ methylene bridge.
 12. Theoligonucleotide of claim 1, wherein the oligonucleotide has a 5′ capstructure, 3′ cap structure, or 5′ and 3′ cap structure.
 13. Theoligonucleotide of claim 1, wherein the oligonucleotide comprises one ormore phosphorothioate linkages.
 14. The oligonucleotide of claim 13,wherein the oligonucleotide is fully phosphorothioate-linked. 15.(canceled)
 16. The oligonucleotide of claim 1, further comprising apendent lipophilic or hydrophilic group.
 17. The oligonucleotide ofclaim 1, wherein the oligonucleotide is about 18 to 50 nucleotides inlength.
 18. (canceled)
 19. The oligonucleotide of claim 1, wherein theoligonucleotide comprises a sequence that is substantially or completelycomplementary to a nucleotide sequence of a mature miRNA.
 20. (canceled)21. The oligonucleotide of claim 1, wherein the miRNA is expressed incardiac tissue.
 22. (canceled)
 23. (canceled)
 24. The oligonucleotide ofclaim 23, wherein the miRNA is not miR208a, miR208b, or miR-499.
 25. Theoligonucleotide of claim 1, wherein the miRNA is miR-15a, miR-15b,miR-16-1, miR-16-2, miR-24, miR-25, miR-26a, miR-497, miR-195, miR-424,a let 7 family member, miR-21, miR-199a-b, miR-214, miR-10a-b, miR-16,miR-125b, miR-146a-b, miR-221, miR-222, a miR-30family member, miR-126,miR-133, miR-1, miR-143, miR-145, miR-486, miR-92a, miR-320, miR-1-1,miR-1-2, miR-451, miR-378, miR-378*, miR-92, miR-34a, miR-34b, miR-34c,miR-29, or miR-33.
 26. The oligonucleotide of claim 6, wherein thesequence is substantially or completely complementary to a nucleotidesequence of an mRNA or mature miRNA.
 27. (canceled)
 28. (canceled)
 29. Apharmaceutical composition comprising an effective amount of theoligonucleotide of claim 1, or a pharmaceutically-acceptable saltthereof, and a pharmaceutically-acceptable carrier or diluent.
 30. Thepharmaceutical composition of claim 29, wherein thepharmaceutically-acceptable carrier comprises a colloidal dispersionsystem, macromolecular complex, nanocapsule, nanoparticle, microsphere,bead, oil-in-water emulsion, micelle, mixed micelle, or liposome. 31.The pharmaceutical composition of claim 29, wherein thepharmaceutically-acceptable carrier or diluent consists essentially ofsaline.
 32. A method of reducing or inhibiting activity of a miRNA in acell comprising contacting the cell with the oligonucleotide of claim 1.33. A method of reducing expression of an mRNA in a cell comprisingcontacting the cell with the oligonucleotide of claim
 6. 34-36.(canceled)
 37. A method of preventing or treating a condition in asubject associated with or mediated by expression of a mRNA, comprisingadministering to the subject a pharmaceutical composition comprising anoligonucleotide of claim
 6. 38. A method of preventing or treating acondition in a subject associated with or mediated by the activity of amiRNA, comprising administering to the subject a pharmaceuticalcomposition comprising the oligonucleotide of claim
 1. 39. The method ofclaim 38, wherein the condition is a heart condition or vascularcondition.
 40. The method of claim 39, wherein the heart condition iscardiac hypertrophy, myocardial infarction, myocardial ischemia, orheart failure.
 41. The method of claim 38, wherein the pharmaceuticalcomposition is administered by intravenous, subcutaneous,intraperitoneal, intramuscular, oral, transdermal, sustained release,controlled release, delayed release, suppository, catheter, orsublingual parenteral administration or by direct injection into cardiactissue. 42-46. (canceled)